Caspase-4 in glioma indicates deterioration and unfavorable prognosis by affecting tumor cell proliferation and immune cell recruitment

Gliomas are the most common malignant tumors of the central nervous system, accounting for approximately 80% of all malignant brain tumors. Accumulating evidence suggest that pyroptosis plays an essential role in the progression of cancer. Unfortunately, the effect of the pyroptosis-related factor caspase-4 (CASP4) on immunotherapy and drug therapy for tumors has not been comprehensively investigated. In this study, we systematically screened six hub genes by pooling differential pyroptosis-related genes in The Cancer Genome Atlas (TCGA) glioma data and the degree of centrality of index-related genes in the protein–protein interaction network. We performed functional and pathway enrichment analyses of the six hub genes to explore their biological functions and potential molecular mechanisms. We then investigated the importance of CASP4 using Kaplan–Meier survival analysis of glioma patients. TCGA and the Chinese Glioma Genome Atlas (CGGA) databases showed that reduced CASP4 expression leads to the potent clinical deterioration of glioma patients. Computational analysis of the effect of CASP4 on the infiltration level and recruitment of glioma immune cells revealed that CASP4 expression was closely associated with a series of tumor-suppressive immune checkpoint molecules, chemokines, and chemokine receptors. We also found that aberrant CASP4 expression correlated with chemotherapeutic drug sensitivity. Finally, analysis at the cellular and tissue levels indicated an increase in CASP4 expression in glioma, and that CASP4 inhibition significantly inhibited the proliferation of glioma cells. Thus, CASP4 is implicated as a new prognostic biomarker for gliomas with the potential to further guide immunotherapy and chemotherapy strategies for glioma patients.

addition, the identification of new markers that synergize with conventional treatment approaches for clinical gliomas represents a promising new therapeutic strategy.
Pyroptosis is a recently discovered form of cell death that occurs through the formation of inflammasomes and activation of the caspase protein cascade.These processes are accompanied by the cleavage of the gasdermin family and release of cell contents including pro-inflammatory factors (e.g., IL1β and IL18) via pores formed in the cell membrane, thereby promoting inflammatory and immune responses 8 .In 2020, Erkes et al. found that BRAF and MEK inhibitors promote T cell infiltration in the immune microenvironment of melanomas through pyroptosis, thereby increasing the sensitivity of the tissue to drug activity and this approach has been widely used in clinical treatment 9 .An increasing number of studies have shown that levels of cellular pyroptosis can be used to predict cancer prognosis, and indicate the occurrence of immune cell infiltration.These studies demonstrate the importance of cellular pyroptosis-related genes in screening for relevant biological targets in the context of radiotherapy, chemotherapy, and immunotherapy for cancers.
Caspase-4 is a member of the caspase family of proteins.The caspase (aspartate-specific cysteine protease) family contains at least 12 members (including CASP1, CASP3, and CASP4) that have a significant impact on cell death, inflammatory responses, the immune microenvironment, and tumor suppression 10 .Caspase-4 (CASP4) has been shown to amplify inflammation and generate cellular pyroptosis in a number of diseases 11,12 .Furthermore, an association between CASP4 levels and outcomes after tumor treatment has been identified.In esophageal squamous cell carcinoma, loss of CASP4 expression has been associated with poor prognosis 13 .However, in epithelial tumors and pancreatic cancer, CASP4 has been shown to play an integral role in promoting tumor cell migration, cell-matrix adhesion and tissue invasion 14 .The specificity of the correlation between CASP4 expression and outcomes in different tumors indicates the potential for synergistic effects with radiotherapy, chemotherapy and immunotherapy in gliomas.
Changes in the tumor microenvironment (TME) are the main cause of tumor progression, and agents that modulate immune cells infiltrating the TME have become critical components of tumor immunotherapy 15 .The glioblastoma microenvironment contains a large number of innate immune cells (e.g., NK cells, and macrophages) 16 , which play important roles in the progression and treatment-resistance of glioma 17 .The TME of glioma has a complex dynamic pattern of communication with tumor cells that is critical for tumor proliferation, migration, and immunosuppression 18 .Furthermore, the inflammatory state of the TME can influence responses to immune checkpoint therapy [19][20][21] .With the widespread clinical use of immune checkpoint blockade (ICB) therapy and its positive impact on treatment outcomes, it has attracted increasing attention in the treatment of glioma 22,23 .
In this study, we screened for a series of genes associated with pyroptosis in gliomas using TCGA data.Through integrated bioinformatics analysis techniques and evaluation of the prognostic significance of pivotal pyroptosis genes, we identified CASP4 as a gene that is critical to glioma pyroptosis.We then used comprehensive analysis and multiple visualization methods to explore the mechanisms by which CASP4 affects the outcomes of glioma therapy.We conducted a series of studies, including analyses of clinical survival prognosis, functional enrichment, and patient staging, to determine the role of CASP4 in gliomas.We also analyzed the correlation between CASP4 expression and glioma immune cell infiltration using The Tumor Immuno Estimation Resource (TIMER) database to explore the potential function of CASP4 in glioma chemotherapy and immunotherapy.Moreover, we experimentally validated the function of CASP4 at the glioma cell and tissue levels.Our findings provide a greater understanding of glioma and new insights into possible strategies for chemotherapy and immunotherapy of glioma.

Data sources and processing
Data (including mRNA expression, mutations) for gliomas up to May 2023 were downloaded from TCGA (https:// portal.gdc.cancer.gov/), a platform containing gene expression profiling data and clinical follow-up information for 530 patients with low-grade glioma (LGG), 167 patients with glioblastoma (GBM), and five neighboring non-tumor tissues.The 22 pyroptosis-related genes differentially expressed between tumor and normal tissues were evaluated using the "Limma" package (version 4.0.2) of R software.The 22 pyroptosis-related genes considered in our study were identified by searching literature published in the previous 3 years (Table 1).Thresholds were set based on the following parameters: cancer versus normal tissue; P-value, 0.05; and a t-test was used to calculate P-values, with P < 0.05 considered to indicate a statistically significant difference.We also used the CGGA (http:// www.cgga.org.cn/) to investigate the expression pattern of CASP4 as well as its clinical and prognostic significance in glioma patients.We collected data for two glioma cohorts (LGG and GBM), including gene expression, clinical information, grade, isocitrate dehydrogenase (IDH) mutation status, 1p/19q code deletion status, and methylation status, as independent test sets.In addition, we recruited patients with gliomas in our clinic to validate our results.

Construction of protein-protein interaction networks
The Search Tool for the Retrieval of Interacting Genes (STRING; https:// string-db.org/) database was used to screen for pyroptosis-related genes in the protein-protein interaction (PPI) network.Without considering disconnected nodes, we used an interaction score > 0.4 to construct the PPI network.Subsequently, the PPI network was visualized using Cytoscape software (version 3.8.2).Pyroptosis-associated genes with degree of centrality ≥ 10 were identified as hub pyroptosis-associated genes using the cytoHubba plugin.

Heatmap analysis
Heatmaps were created using TCGA expression profiles and visualized using a heatmap software package.

Survival prognosis analysis
Based on TCGA data, Gene Expression Profiling Interactive Analysis 2 (GEPIA2; http:// gepia.cancer-pku.cn/) was used to investigate the overall survival (OS) and disease-free survival (DFS) associated with the six hub pyroptosis-related genes in gliomas.Based on the median value for gene expression, we plotted Kaplan-Meier survival curves after dividing all samples into high and low CASP4 expression groups.Analyses were performed using risk ratios (95% confidence intervals) and log-rank tests.P < 0.05 was considered to indicate statistical significance.

TIMER database analysis
To determine the relationship between CASP4 expression and cellular pyroptosis in gliomas, nine genes (CASP1, CASP3, CASP5, GSDMD, AIM, NLRP1, NLRP3, IL-1B, and IL-18) related to the classical pyroptosis pathway were analyzed for their relevance to gliomas (LGG + GBM) based on data obtained from TCGA.P-values were calculated for each sample, and P < 0.05 was considered to indicate statistical significance.The Tumor Immuno Estimation Resource (TIMER; https:// cistr ome.shiny apps.io/ timer/), which is a comprehensive web server and includes more than 10,000 samples from multiple cancer types in TCGA, was used to determine the correlation between CASP4 expression and the abundance of classically pyroptotic cells in gliomas.To determine the correlations of CASP4 expression with immunosuppressant use and chemokine and chemokine receptor expression in human gliomas, we used data from the Tumor and Immune System Interaction Database (TISIDB; http:// cis.hku.hk/ TISIDB).Based on data obtained from TCGA, we analyzed the correlations of CASP4 expression with immunosuppressant use and chemokine and chemokine receptor expression in LGG and GBM using Spearman analysis.P < 0.05 was considered to indicate statistical significance.

Glioma clinical information analysis
To further explore the relationship between CASP4 expression and the clinicopathological characteristics of glioma patients, we used patients' age, gender, molecular subtype, pathological grade, clinical stage, IDH mutation status, 1p/19q code deletion status, and methylation status as categorical variables.

Correlation analysis of IC50
We explored the correlation between CASP4 and glioma sensitivity to various chemotherapeutic agents using data from the Genomics of Drug Sensitivity in Cancer (GDSC; http:// www.cance rrxge ne.org) database and Pearson analysis.

Transfection of cultured cells
Cultured cells were transfected with plasmid DNA using INTERFERin transfection reagent (101,000,028, Polyplus-transfection, Illkirch, France) according to the manufacturer's instructions.After 48 h, the cells were harvested for Western blotting.The CASP4 siRNA sequence was GUG UAG AUG UAG AAGAAtt.

Colony formation assay
U251 cells in culture medium containing 10% FBS and 1% antibiotics were seeded into 6-well plates.For siRNA treatment, 2 μg of siRNA mixed with the same volume of transfection reagent (Polyplus-transfection, Illkirch, France) was added to the cells and incubated for 6 h.The cells were then centrifuged at 800 rpm for 3 min at room temperature, washed three times with PBS, and seeded into 6-well plates (500 cells per well).All cells were cultured at 37 °C under 5% CO 2 for 10 days and then stained with crystal violet.

Western blot analysis
Cells were lysed with RIPA buffer, and total protein was extracted.Protein concentration was determined using a BCA protein assay kit (P0011, Beyotime, Shanghai, China) according to the manufacturer's instructions.Subsequently, proteins were separated by 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to membranes.The corresponding proteins were detected using antibodies against CASP4 (1:1000; WL04243, Wanleibio, Shenyang, China) and GAPDH ( www.nature.com/scientificreports/Danvers, MA, USA).Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000, ZSGB-BIO, Beijing, China) 24 .Photographs were obtained using a fluorescence/chemiluminescence imaging system (CLINX Scientific Instruments, Shanghai, China) and analyzed using ImageJ software.Each experiment was repeated at least three times.

Immunohistochemical staining
Tissues from three glioma patients were fixed with 10% formaldehyde and embedded in paraffin.Subsequently, the tissues were sectioned and incubated overnight at 4 °C with an anti-CASP4 antibody (1:100; WL04243, Wanleibio, Shenyang, China).After washing with PBS, sections were incubated with HRP-labeled goat anti-rabbit secondary antibody (1:200) for 30 min at room temperature.The samples were washed with PBS, incubated with DAB for 2-5 min at room temperature, then stained with hematoxylin.Micrographs of five unduplicated areas were obtained using an Olympus microscope.

Statistical analysis
Statistical analyses were performed using ImageJ software (1.38e) and the Statistical Package for the Social Sciences (SPSS 26.0).Results were visualized using GraphPad Prism 9.2.0.Correlations were evaluated using Pearson's or Spearman's analyses.Student's t-test was used to determine statistical significance of differences between groups.Differences between more than two groups were analyzed using one-way ANOVA.All experiments were repeated three times for validation.P-values < 0.05 were considered to indicate statistical significance.

Ethical approval and consent to participate
This study was approved by the Institutional Review Board of Harbin Medical University (Approval No. KY2023-072; Harbin, China), and the study was conducted in accordance with the Declaration of Helsinki.All participants provided written informed consents to participate in this study.

Screening and identification of pyroptosis-related genes in gliomas
The process used to screen pyroptosis-related genes in gliomas is described in Fig. 1.From TCGA, we carefully screened and downloaded data on a total of 530 LGG and 167 GBM tumors and non-tumor samples for comparison.Details of the 22 possible pyroptosis-related genes that have been studied in tumor patients in recent years are shown in Table 1.A total of 11 genes associated with pyroptosis were found to be differentially expressed in glioma tumors and adjacent non-tumor tissues (Fig. 2A,B; P < 0.05).We used Cytoscape to visualize the relationship between the two groups of significantly differentially expressed genes on a PPI network graph (Fig. 2C, yellow: P < 0.05; blue: ns, P > 0.05).Then, to further screen for pyroptosis-related genes that play essential roles in the pathogenesis of gliomas, we performed detailed analysis of the 22 possible pyroptosis-related genes using the STRING database and sorted the pivotal genes by degree centrality using the Cytoscape plugin cytoHubba.

Biological functions of the screened genes are closely related to cancer development
To explore the related pathways and functions, we performed GO and KEGG enrichment analysis of the six hub genes.These genes were enriched mainly in immune, pyroptosis, and tumor-related biological processes, such as cytokine-mediated signaling pathways, responses to cytokines, responses to tumor necrosis factor, pyroptosis processes, interleukin (IL)-1β production, T cell proliferation, B cell activation, T cell activation, leukocyte homeostasis, and activation of T cells involved in the immune response (Fig. 3A).Cellular components associated with the hub genes were associated with tumor cell pyroptosis and proliferation, and cellular localizations included mitochondria, inflammasome complex, plasma membrane protein complex, membrane protein complex, peptidase inhibitor complex, death-inducing signaling complex, replication forks, and double-strand breakpoints (Fig. 3A).In addition, molecular functional analysis verified that the six hub genes were enriched mainly in cysteine-type peptidase activity, endopeptidase activity, protein domain-specific binding, protease binding, cytokine receptor binding, death receptor binding, aspartate-type peptidase activity, and disordered domain-specific binding (Fig. 3A).KEGG pathway enrichment analysis showed that the six pyroptosis-related hub genes were enriched mainly in the Nucleotide-binding Oligomerization Domain (NOD)-like receptor, cellular DNA sensing, p53, IL-17, C-type lectin receptor, and Tumor Necrosis Factor (TNF) signaling pathways, as well as platinum resistance, colorectal cancer, and small cell lung cancer (Fig. 3B).Functional analyses suggested that the six hub genes may be intimately involved in tumor cell pyroptosis, drug resistance among tumor cells, regulation of the tumor immune microenvironment, and tumor development.

High CASP4 expression significantly correlates with the poor prognosis of glioma patients
To assess the role of the pyroptosis-related hub genes in independent prognoses, we extracted clinical information on a cohort of glioma patients from TCGA.Based on the threshold of the median risk score, we allocated the glioma patients to high-and low-risk groups.Kaplan-Meier survival curves constructed to differentiate the OS and DFS of the glioma patients showed that CASP4 was strongly associated with OS in glioma patients (P < 0.0001, Fig. 4A).The correlation between the upregulation of CASP4 expression levels and poor prognosis of glioma patients was the most significant compared to changes in the other pivotal pyroptosis-related genes.Similar results were observed with corresponding DFS data (P < 0.0001, Fig. 4B).These findings implicate CASP4 as a prospective biomarker for gliomas.Although some reports have addressed the role of CASP4 in gliomas, to the best of our knowledge, the mechanisms underlying the role of CASP4 in immunotherapy and chemotherapy have not been fully elucidated; therefore, we selected CASP4 for further analysis.
T N F T P 5 3 T P 6 3

CASP4 expression is strongly associated with pyroptosis genes of patients with glioma
We first analyzed the relationship between CASP4 expression and glioma cell pyroptosis as a recently discovered form of cell death.Recent studies have shown that the caspase family triggers cellular pyroptosis through the cleavage of GSDMD protein, which releases the corresponding inflammatory factors via a pathway involving nine related proteins: CASP1, CASP3, CASP5, GSDMD, AIM2, NLRP1, NLRP3, IL-1B, and IL-18 25,26 .According to the data obtained from TCGA, eight classical pyroptosis pathway-related genes were found to be associated with the development of gliomas (LGG + GBM) (Fig. 5A).The PPI network of the proteins encoded by these eight classical pyroptosis genes and CASP4 revealed eight CASP4 node edges (Fig. 5B), indicating the importance of CASP4 in weighting the relationships among the nine pyroptosis-related hub genes.The TIMER web server was further employed to determine the correlation between CASP4 expression with the eight classical pyroptosis genes in GBM and LGG.We found that CASP4 expression was positively correlated with the eight classical pyroptosis genes in both LGG and GBM (Fig. 5C,D).

Aberrant overexpression of CASP4 is correlated to adverse clinicopathological features of glioma
The gene expression profiles of all glioma samples and paired normal tissues showed that CASP4 was overexpressed in gliomas.In recent years, 1p/19q coding deletions, IDH mutations, and methylation of the O-6-methylguanine DNA methyltransferase (MGMT) gene promoter have been widely used in the diagnosis and prognostic assessment of gliomas 27 .To further explore the relationship between high CASP4 expression and the clinical presentation of glioma patients, we investigated the relationship between CASP4 expression and the characteristics of age, gender, molecular subtype, pathological grade, clinical stage, isocitrate dehydrogenase (IDH) mutation, 1p/19q codeletion, and MGMT methylation status.Graphical representation of these characteristics confirmed that CASP4 overexpression in the TCGA dataset was significantly correlated with WHO grade, IDH genotype, 1p/19q code, MGMT gene promoter methylation status, and age.Furthermore, we found that CASP4 expression was significantly lower in the IDH mutation group, the 1p/19q chromosome group, and MGMT gene promoter methylation group, and increased significantly with WHO grade and age (Fig. 6A-E).CASP4 expression also varied significantly among different histological subtypes (Fig. 6G-I), whereas there was no difference in CASP4 expression between male and female patients (Fig. 6F).These results were validated in similar evaluations of these characteristics for glioma patients in the CGGA database (Fig. 6J-P).These findings suggested that CASP4 is significantly overexpressed in gliomas and is associated with adverse clinicopathologic features.
To explore the correlation between CASP4 expression and the proportions of infiltrating immune cells, we assessed the status of 22 tumor-infiltrating immune cell types using CIBERSORT analysis.We found that the proportion of macrophages was significantly higher than that of the other immune cells (Fig. 7B).Correlation analysis showed that 18 infiltrating cell types were significantly associated with CASP4 expression (P < 0.01).As shown in Fig. 7C, memory B cells, resting NK cells, macrophages, memory activated CD4 + T cells, memory resting CD4 + T cells, activated dendritic cells, resting mast cells, neutrophils, and regulatory T cells (Tregs) were positively correlated with CASP4 expression, while naïve B cells, plasma cells, naive CD4 + T cells, follicular helper T cells, activated NK cells, monocytes, and activated mast cells were negatively correlated with CASP4 expression.We also used the ESTIMATE, immunity, and stroma scores to assess the links between CASP4 and the glioma immune microenvironment.Figures 7D,E show that high CASP4 expression was linked to higher ESTI-MATE, immunity, and stromal scores, but lower glioma purity (P < 0.0001).In addition, we employed the TIP database to analyze the relationship between CASP4 and different aspects of tumor immunotherapy processes.Figures 7F,G show the seven-step cycle underlying the involvement of CASP4 in anticancer immunity in gliomas: Step 1, cancer cell antigen release; Step 2, cancer antigen presentation; Step 3, induction and activation; Step 4, migration of immune cells toward the tumor; Step 5, immune cell infiltration of the tumor; Step 6, recognition of the cancer cell by T cells; and Step 7, killing of the cancer cell.The data showed that high CASP4 expression is strongly correlated with the degree of migration and recruitment of immune cells to the tumor (Step 4), thus affecting the anticancer immune status and the proportion of tumor-infiltrating immune cells in glioma patients.

Validation of CASP4 expression and function in cells and tissues of glioma
To confirm the expression of CASP4 at the cancer cell and tissue level, we collected glioma cells and histopathological sections from patients.In accordance with the results from TCGA data, our analysis showed that CASP4 expression was elevated in glioma cells compared with normal astrocytes, and CASP4 was highly expressed in gliomas compared with normal paracancerous tissues (Fig. 10A-D).To further validate the effect of CASP4 on glioma cells, we inhibited CASP4 expression in U251 glioma cells and verified the inhibition efficiency by Western blot (Fig. 10E,F).Colony formation assays showed that inhibition of CASP4 expression significantly suppressed cell proliferation (Fig. 10G,H).Finally, GSEA confirmed the involvement CASP4 upregulation in pathways related to tumor cellular pyroptosis and immunity (Fig. 10I).Therefore, our comprehensive analyses indicated that CASP4 is closely associated with cellular pyroptosis, immunotherapy, and drug treatment of gliomas, suggesting the potential of CASP4 as a novel therapeutic target in gliomas.

Discussion
Gliomas are the most common malignant brain tumors, and despite the evolution of traditional treatments such as radiotherapy chemotherapy and immunotherapy, their prognosis has changed little over the past few decades.The invasiveness, recurrence, and drug resistance of gliomas are the main reasons for the poor prognosis of patients 31,32 .Therefore, exploring the mechanisms and useful targets associated with improved immunotherapy and chemotherapy of glioma patients is the focus of contemporary neurological research.Pyroptosis is an emerging mode of cell death that may have a dual role in gliomas 33 .Risk models composed of genes or long non-coding RNAs related to pyroptosis have shown good predictive and diagnostic value in gliomas, although the exploration of key genes and therapeutic targets of pyroptosis has not been comprehensive, and there is a lack experimental evidence 34,35 .In our study, we targeted the key gene CASP4 in a differential analysis of pyroptosis-related genes in gliomas and their relationship to clinical prognosis.Relying on comprehensive bioinformatics analyses, we demonstrated that differences in the degree of CASP4 expression are strongly associated with the prognosis of patients with gliomas.CASP4 expression may serve as an independent prognostic marker, as it correlates with a variety of clinical features, particularly the pathological stage and status of the tumor.In relation to the poor outcomes of immunotherapy and drug therapy in glioma patients, we found that glioma CASP4 expression correlated with the degree of infiltration of multiple immune cell types and sensitivity to chemotherapeutic agents.These findings suggest that CASP4 expression affects the efficacy of immunotherapy and chemotherapy of gliomas and implicate CASP4 is a promising therapeutic target.Based on recent data from TCGA, we analyzed 22 differentially expressed pyroptosis-related genes in glioma patients, and then combined the results of two protein interactions analyses to identify six key pyroptosis-related genes in gliomas.To gain a more in-depth and systematic understanding of the biological roles of these genes, we performed GO and KEGG analyses.GO analysis revealed that the functions of the six pivotal pyroptosis genes are enriched mainly in tumor immunity, cellular pyroptosis, and other tumor-related biological processes and are closely related to glioma development, including cytokine-mediated signaling pathways that can directly affect the progression of malignant gliomas.Cytokines maintain the precise balance between glioma cells and the TME, thus contributing to the synergistic immunological and pharmacological treatment of gliomas 36 .Cellular localization analysis revealed that some of the six pivotal pyroptosis genes were localized in sites such mitochondria and the inflammasome complex.In the clinic, both mitochondrial transplantation and anti-inflammatory complexes are highly promising therapeutic modalities for gliomas, which may indicate an important function of pyroptosis genes in glioma therapy 37,38 .KEGG pathway enrichment analysis showed that the six pivotal pyroptosis genes were enriched mainly in the NOD-like receptor and p53 signaling pathways and platinum resistance, the roles of which in gliomas have been widely confirmed.The NOD-like receptor signaling pathway is central to the pathogenesis of many cancers and neurodegenerative diseases, and NOD-like receptor signaling contributes to the angiogenesis of gliomas, with NLRP3 being an important component, which promotes the growth and invasion of gliomas through IL-1β/NF-κB p65 signaling 39,40 .It has been suggested that the p14ARF-MDM2-p53 pathway is one of the major pathways in the molecular genetic pathogenesis of gliomas, in which p14ARF and MDM2 proteins are involved in glioma development by regulating the ubiquitination-mediated degradation of p53.As one of the most important oncogenic proteins, mutations in p53 can lead to the aberrant proliferation of tumor cells and promote the deterioration of cerebral glioma 41 .All of these gene function analyses indicate that the six key genes are involved in tumor cell pyroptosis, tumor immune microenvironment regulation, and tumor development through multiple pathways.Using the online database GEPIA2, we found that the correlation between CASP4 expression levels and poor glioma prognosis (OS and DFS) was the most significant, suggesting that CASP4 plays a critical role in the prognosis of glioma treatment.To the best of our knowledge, the current literature on the role of CASP4 in glioma immunotherapy and chemotherapy is superficial and limited in scope; therefore, we chose to conduct an in-depth and comprehensive analysis of the role of CASP4 in the treatment of glioma.
CASP4 plays a key role in a variety of diseases such as spinal cord injury and Alzheimer's disease-related synaptic and behavioral deficits, and its role in tumors seems to be two-fold 13,[42][43][44] .We explored the relationship between CASP4 and tumor behavior, tumor subtype, and other clinical features of gliomas.By exploring TCGA and CGGA, we found that CASP4 was significantly associated with WHO class, IDH genotype, 1p/19q coding, MGMT gene promoter methylation, and age.1p/19q gene deletion, IDH gene mutation and MGMT gene promoter methylation have been widely studied and identified as diagnostic and predictive markers for glioma typing and tumor progression 45 .CASP4 may guide the development of different types of gliomas, and it is expected to make an important contribution to the prediction of glioma typing and the development of individualized treatments.One of the main reasons for the individual variability in the effects of immunotherapy on the prognosis of patients with glioma may be differences in the proportions of immune cells in the tumor infiltrate.Therefore, we used various immunoassay databases, such as TIMER, CIBERSORT, ESTIMATE, and TIP, to determine the type of immune cell infiltration that is most closely associated with CASP4.Although the TIMER immunoassay database showed that CASP4 is closely associated with dendritic cells in glioma patients, the percentage of dendritic cells in glioma immune cells, as revealed by the CIBERSORT algorithm, is very small.Therefore, dendritic cells, although closely associated with CASP4 expression, may not be the main reason why CASP4 affects immunotherapy in gliomas.Using the CIBERSORT algorithm, we found that M2 type macrophages were not only present at a high percentage, but also exhibited greater infiltration of gliomas expressing high levels of CASP4.Macrophages can promote tumor development by regulating tumor cell metabolism and angiogenesis 46 .As one of the major regulatory components in the TME, the role of tumor-associated macrophages (TAMs) in gliomas has attracted much attention because they are significantly associated with glioma progression, grading, and prognosis.Pleiotrophin secretion by TAM promotes signaling in glioblastoma stem cells and tumor growth 47 .TAMs can be functionally subtyped according to their polarization status (i.e., M1 and M2), and several studies have demonstrated a pro-tumorigenic role for M2 TAMs in gliomas 48 .Preventing TAM polarization to the M2 subtype has been reported to block glioma progression and tumor growth 49 .Our study showed that CASP4 expression followed the same trend as macrophage M2 infiltration, suggesting that CASP4 may have an impact on the associated immune cell infiltration, thereby affecting the susceptibility of glioma patients to immunotherapy.Furthermore, previous studies have shown that the expression of some combinations associated with immune steps is also a prognostic predictor for glioblastoma, e.g., the combined expression of CD276, GATA3, and LGALS3, as well as the expression of PD-L1, PD-L2, and PD-1 predicted the prognosis of glioma patients 50,51 .Based on the critical role of immune scores and steps in glioma treatment, our study identified a strong correlation between CASP4 expression and ESTIMATE scores and immune steps in glioma patients.In ESTIMATE and TIP immunoassay analyses, CASP4 expression significantly affected ESTIMATE scores and anticancer immune steps, including cancer cell antigen release, cancer antigen presentation, induction and activation, immune cell transfer to the tumor, immune cell infiltration of the tumor, T cell recognition of the cancer cells, and killing of the cancer cells 52 .Immunologic drugs affect the course of immunotherapy by targeting these seven steps.Investigating the role of CASP4 in influencing these steps has the potential to improve the efficacy of immunotherapy for glioma, making CASP4 an important predictor of accurate glioma treatment outcomes.
Considering the important role of chemokines in immune cell migration, we further explored whether the role of CASP4 in the immune microenvironment of gliomas is related to this type of regulatory factors.As an immune checkpoint receptor, CD96 has been extensively demonstrated to have biological functions in T cells and natural killer cells 53 , playing an essential role in immunotherapy and the poor prognosis of many cancers, including hepatocellular carcinomas and gliomas 54,55 .CSF1R regulates macrophage activation, proliferation, and phagocytosis mainly in the tumor immune microenvironment 56 .CSF1R inhibition alters macrophage polarization, thereby blocking glioma progression, and has been identified as a highly promising therapeutic method 49 .Similarly, CCL2/CCR2 affects the recruitment of TAMs, which in turn influences prognosis 57 .CCL5/CCR5, on the other hand, affects the efficacy of tumor therapy mainly by promoting inflammatory responses as well as inducing the adherence and migration of different T cell subsets involved in the immune response 58 .In this study, using TISIDB and correlation analysis, we identified a close correlation between CASP4 and the abovementioned regulators, in addition to positive correlations between CASP4 and HAVCR2, IL10, CXCL8, CCR5, CCR7, CXCR2, and other immune cell-associated regulators.To further illustrate how CASP4 affects many of the above factors and thus, plays a key role in gliomas, we performed functional interplay analysis and identified the key molecule IL-1β using PPI network analysis.IL-1β is an important downstream factor that exerts the pro-inflammatory effects of pyroptosis 18 .Numerous studies have demonstrated the crucial role of IL-1β in T cell polarization, acute inflammation, and adaptive anti-tumor responses; whereas, in chronic inflammatory processes, the IL-1β produced by tumor-infiltrating macrophages may play a supportive role in tumor development 59,60 .The above results suggest that CASP4 influences the activation, proliferation, and infiltration of immune cells via the interaction of IL-1β with various immunomodulatory factors, chemokines, and their receptors, to affect the efficacy of immunotherapy for gliomas.
Drug therapy is the conventional approach to tumor treatment, and decreased drug sensitivity is an important factor that affects the progression of most tumors.Due to the limitations of radiotherapy for the treatment of neurological tumors, many studies have focused on improving the sensitivity of glioma cells to chemotherapeutic agents such as paclitaxel 61 .For this reason, we investigated the correlation between CASP4 and chemotherapeutic drug sensitivity with the aim of providing possible targets for improving the efficacy of chemotherapy in glioma patients.Paclitaxel is regarded as a classical antitumor drug because of its unique mechanism of action, although the incidence of tumor resistance is becoming increasingly widespread and is found in a diversity of cancers, such as triple-negative breast cancer and lung cancer 62,63 .The issue of decreased sensitivity is also inevitable in the clinical application of sunitinib.Jiang et al. suggested that elevated SNRPA1 predicts the sensitivity of clear cell renal cell carcinoma to sunitinib 64 .Based on our study, we propose that the important pyroptosis gene CASP4 can be used to predict the sensitivity of glioma cells to paclitaxel and sunitinib.In addition, we found sensitivities to several other well-known antitumor drugs, such as dabrafenib, dasatinib, rapamycin, that are also closely related to CASP4 expression.Further studies on the specific mechanisms of CASP4 expression and drug resistance could provide new ideas for future chemotherapy development.
Finally, we verified the aberrant expression of CASP4 by in vitro analysis of cells and histological samples from glioma patients.Furthermore, we showed that CASP4 expression affected glioma cell proliferation.Finally, the results of GSEA indicated that CASP4 is involved in the glioma-associated NLRP3-inflammation complex as well as immune signaling pathways such as T cell activation and proliferation and chemokine activation.These pathways affect tumor immunotherapy, drug therapy, and patient prognosis in gliomas, and CASP4 has demonstrated its potential as a prognostic predictor and therapeutic target.

Conclusion
CASP4 is a predictive therapeutic target that is closely related to immunotherapy and resistance to chemotherapeutic agents in gliomas.Our comprehensive and detailed analysis suggests that CASP4 may improve the prognosis of glioma patients mainly by affecting the immune microenvironment of the tumor, and the reduction of its expression level is an important guideline for improving the efficacy of immunotherapy and chemotherapy in glioma patients with different phenotypes and at different stages.

Figure 1 .
Figure 1.Flow diagram of study procedure.

Figure 2 .
Figure 2. Expression and interaction network of pyroptosis-related genes in gliomas.

Figure 10 .
Figure 10.Validation of CASP4 expression and function in glioma.(A) CASP4 expression in normal tissue and glioma tissue.Scale bar, 100 μm.(B) Quantification of CASP4 protein levels in normal tissue and tumor tissue, Data represent the mean ± SD, n = 3. (C) CASP4 expression in astrocytes HA1800 and glioma cells U251 detected using Western blot analysis; GAPDH was used as an internal control.(D) Quantification of CASP4 protein levels in HA1800 and U251 cell lines.Data represent the mean ± SD, n = 3. (E) Efficiency of CASP4 inhibition mediated by siRNA detected by Western blot; GAPDH was used as an internal control.(F) Quantification of CASP4 protein inhibition mediated by siRNA.Data represent the mean ± SD, n = 3. (G) CASP4 inhibition significantly inhibited U251 cell migration as determined by colony formation assay.(H) Quantification of the number of colonies.Data represent the mean ± SD, n = 3. (I) Important signaling pathways associated with CASP4 according to GSEA.

Table 1 .
Details of the 22 possible pyroptosis-related genes that have been studied in tumor patients in recent years.